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PubMed2004

Induction of Cdc25B expression by epidermal growth factor and transforming growth factor-alpha.

The dual specificity protein phosphatase Cdc25B regulates of the mitotic cell cycle checkpoint and is over expressed in human tumors. Given the importance of growth factors in initiating and sustaining cell proliferation, we examined their effects on Cdc25B protein expression in human cancer cells. Within 1h after epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) treatment, Cdc25B protein levels increased in growth factor responsive A549 and SCC25 cells, but not in non-responsive MDA-MB-231 cells. A functional consequence of elevated Cdc25B was implied by the concomitant decrease in phosphorylated cyclin dependent kinase, a known Cdc25B substrate, after growth factor treatment of A549 and SCC25 cells. The EGF-mediated induction of Cdc25B required a functional EGF receptor (ErbB1), as mouse embryonic fibroblasts lacking ErbB1 did not have increased Cdc25B levels after EGF treatment. Moreover, the EGFR receptor-selective tyrosine kinase inhibitor AG1478 and mitogen activated kinase kinase inhibitor U0126 blocked growth factor-mediated Cdc25B induction. Thus, EGF and TGF-alpha appear to induce cellular Cdc25B through the mitogen-activated protein kinase pathway.

Butadienes↗
PubMed2005

Control of cell proliferation in the Drosophila eye by Notch signaling.

Cell proliferation in animals must be precisely controlled, but the signaling mechanisms that regulate the cell cycle are not well characterized. A regulated terminal mitosis, called the second mitotic wave (SMW), occurs during Drosophila eye development, providing a model for the genetic analysis of proliferation control. We report a cell cycle checkpoint at the G1-S transition that initiates the SMW, and we demonstrate that Notch signaling is required for cells to overcome this checkpoint. Notch triggers the onset of proliferation by multiple pathways, including the activation of dE2F1, a member of the E2F transcription factor family. Delta to Notch signaling derepresses the inhibition of dE2F1 by RBF, and Delta expression depends on the secreted proteins Hedgehog and Dpp. Notch is also required for the expression of Cyclin A in the SMW.

Animals↗
PubMed2000

The murine Pes1 gene encodes a nuclear protein containing a BRCT domain.

Pescadillo was originally identified in the zebrafish Danio rerio as a site of a retrovirus-insertion mutation that caused severe defects during embryogenesis. In particular, growth of the fetal zebrafish liver was significantly affected by loss of pescadillo function. To begin to understand the role of pescadillo during mammalian hepatogenesis we identified the murine homologue of pescadillo and named it Pes1. A single gene localized to chromosome 11 on the mouse genome encodes Pes1. Although Pes1 mRNA was detected in all tissues examined it was present at the highest levels in both adult and fetal liver. Analysis of the predicted amino acid sequence of Pes1 found it to contain a BRCT domain, which has previously been found in several proteins involved in cell-cycle checkpoints and DNA repair. Consistent with a putative role in these processes we found that when recombinant Pes1 protein was expressed in HepG2 cells it localized to the nucleus.

Amino Acid Sequence↗
PubMed2002

Expression of the chicken homologue of the mouse double minute 2 gene.

While the p53 tumor suppressor plays a crucial role in regulating cell cycle checkpoints and apoptosis by acting as either a transcriptional activator or repressor in a variety of mammalian cells, its evolutionarily conserved functions remain to be elucidated in non-mammalian species. In the present study, we determined the functional role of p53 in avian cells by analyzing the expression pattern of the chicken homologue (CDM2) of mouse double minute 2, one of the transcriptional target genes of p53. CDM2 displayed considerable conservation in the p53 binding region as well as the nuclear localization and nuclear export signals and was found to be abundantly expressed in the reproductive organs (testis and ovarian follicles) and in the immune organs (bone marrow, bursa and thymus). CDM2 expression exhibited an early serum-response pattern consistent with its mammalian counterparts and was dramatically downregulated in most of the p53-downregulated immortal chicken embryo fibroblast (CEF) cells analyzed. Expression of CDM2 was shown to be transcriptionally upregulated in the primary CEF cells where p53 was activated by either mitomycin C treatment or by the exogenous transfection of the chicken p53 cDNA. Together, the current studies demonstrate that the expression of MDM2 homologues may be biologically conserved in mammalian and avian cells.

Amino Acid Sequence↗
PubMed2002

Additive roles of XPA and MSH2 genes in UVB-induced skin tumorigenesis in mice.

We have made xeroderma pigmentosum group A gene (XPA)-knockout mice (XPA(-/-) mice). The XPA(-/-) mice had no detectable activity for nucleotide excision repair (NER) and showed a high incidence of UVB-induced skin tumorigenesis. We have also found that cell lines derived from skin cancers in UVB-irradiated XPA(-/-) mice become tolerant to UV-irradiation and showed abnormal UV-induced cell cycle checkpoints and decreased mismatch repair (MMR) activity. These results suggested that the MMR-downregulation may help cells escape killing by UV-irradiation and thus MMR-deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells. In this report, we examined whether the incidence of UVB-induced skin tumorigenesis is enhanced in XPA(-/-)MSH2(-/-), XPA(-/-) and MSH2(-/-) mice when compared with that in wild-type mice. Our results indicate that the MSH2-deficiency caused a high incidence of spontaneous and UVB-induced skin tumorigenesis and the XPA and MSH2 genes have additive roles in the UV-induced skin tumorigenesis.

Animals↗
PubMed2005

DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system.

Cell-cycle checkpoints induced by DNA damage or replication play critical roles in the maintenance of genomic integrity during cell proliferation. Biochemical analysis of checkpoint pathways has been greatly facilitated by the use of cell-free systems made from Xenopus eggs. In the present study, we describe a human cell-free system that reproduces a DNA-dependent checkpoint pathway acting on the Chk1 protein kinase. In this system, double-stranded DNA oligonucleotides induce the phosphorylation of Chk1 at activating sites targeted by ATR [ATM (ataxia telangiectasia mutated)- and Rad3-related] and ATM kinases. Phosphorylation of Chk1 is dependent on the interaction of Claspin, a protein first identified in Xenopus as a Chk1-binding protein. We show that the DNA-dependent binding of Chk1 to Claspin requires two phosphorylation sites, Thr916 and Ser945, which lie within the Chk1-binding domain of Claspin. Using a phosphopeptide derived from the consensus motif of these sites, we show that the interaction of Claspin with Chk1 is required for the ATR/ATM-dependent phosphorylation of Chk1. Using a panel of protein kinase inhibitors, we provide evidence that Chk1 is phosphorylated at an additional site in response to activation of the checkpoint response, probably by autophosphorylation. Claspin is phosphorylated in the Chk1-binding domain in an ATR/ATM-dependent manner and is also targeted by additional kinases in response to double-stranded DNA oligonucleotides. This cell-free system will facilitate further biochemical analysis of the Chk1 pathway in humans.

Adaptor Proteins, Signal Transducing↗
PubMed2000

p53 regulates myogenesis by triggering the differentiation activity of pRb.

The p53 oncosuppressor protein regulates cell cycle checkpoints and apoptosis, but increasing evidence also indicates its involvement in differentiation and development. We had previously demonstrated that in the presence of differentiation-promoting stimuli, p53-defective myoblasts exit from the cell cycle but do not differentiate into myocytes and myotubes. To identify the pathways through which p53 contributes to skeletal muscle differentiation, we have analyzed the expression of a series of genes regulated during myogenesis in parental and dominant-negative p53 (dnp53)-expressing C2C12 myoblasts. We found that in dnp53-expressing C2C12 cells, as well as in p53(-/-) primary myoblasts, pRb is hypophosphorylated and proliferation stops. However, these cells do not upregulate pRb and have reduced MyoD activity. The transduction of exogenous TP53 or Rb genes in p53-defective myoblasts rescues MyoD activity and differentiation potential. Additionally, in vivo studies on the Rb promoter demonstrate that p53 regulates the Rb gene expression at transcriptional level through a p53-binding site. Therefore, here we show that p53 regulates myoblast differentiation by means of pRb without affecting its cell cycle-related functions.

Animals↗
PubMed2005

Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress.

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.

Apoptosis↗
PubMed2000

Activation of the transcription factor Oct-1 in response to DNA damage.

Mammalian cells exhibit complex cellular responses to genotoxic stress, including cell cycle checkpoint, DNA repair, and apoptosis. Inactivation of these important biological events will result in genomic instability and cell transformation. It has been demonstrated that gene activation is a critical initial step during the cellular response to DNA damage. A number of investigations have shown that transcription factors are involved in the regulation of stress-inducible genes. These transcription factors include p53, c-Myc, and AP-1 (c-fos and c-jun). However, the role for the octamer-binding transcription factor Oct-1 in the DNA damage-activated response is unknown. In this report, we have presented the novel observation that the transcription factor Oct-1 is induced after cells are exposed to multiple DNA-damaging agents and therapeutic agents, including UV radiation, methylmethane sulfonate, ionizing radiation, etoposide, cisplatin, and camptothecin. The induction of the Oct-1 protein is mediated through a posttranscriptional mechanism and does not require the normal cellular function of the tumor suppressor p53, indicating that the Oct-1 protein, as a transcription factor, may play a role in p53-independent gene activation. In addition to increased protein level, the activity of Oct-1 DNA binding to its specific consensus sequence is also enhanced by DNA damage. Therefore, these results have implicated that the transcription factor Oct-1 might participate in cellular response to DNA damage, particularly in p53-independent gene activation.

DNA Damage↗
PubMed2005

Roscovitine modulates DNA repair and senescence: implications for combination chemotherapy.

PURPOSE: Treatment of tumor cells by chemotherapy activates a series of responses ranging from apoptosis to premature senescence and repair. Survival responses are characterized by inhibition of cyclin-dependent kinases. Because inhibition of cyclin-dependent kinases represents a distinctive feature of DNA damage-induced prosurvival responses, we investigated the possibility that the cyclin-dependent kinase inhibitor roscovitine modulates drug-induced responses in human adenocarcinoma cells, favoring cell survival. EXPERIMENTAL DESIGN: Sublethal concentrations of doxorubicin were used to induce premature senescence in human adenocarcinoma cells. The effect of the cyclin-dependent kinase inhibitor roscovitine on the doxorubicin-dependent cell cycle checkpoint activation and DNA repair pathways was evaluated. RESULTS: Roscovitine reinforces doxorubicin-dependent G(1) checkpoint in A549 and HEC1B cells leading to decreased frequency of double-strand breaks and to the preferential induction of senescence and enhanced clonogenic survival. However, in other tumor cell lines, such as HCT116 and H1299, combined treatment with doxorubicin and roscovitine increases the frequency of double-strand breaks and dramatically sensitizes to doxorubicin. This unexpected effect of roscovitine depends on a novel ability to inhibit DNA double-strand break repair processes and requires inactivation of the pRb pathway. CONCLUSIONS: Roscovitine, by hindering DNA repair processes, has the potential to inhibit recovery of mildly damaged tumor cells after doxorubicin treatment and to increase the susceptibility of tumor cells to chemotherapy. However, in some tumor cells, the cell cycle inhibitory function of roscovitine prevails over the DNA repair inhibitory activity, favoring premature senescence and clonogenic growth. These data indicate a novel mechanism underlying combined chemotherapy, which may have wide application in treatment of carcinomas.

Antineoplastic Agents↗
PubMed1998

Involvement of p16CDKN2A in cell cycle delays after low dose UV irradiation.

Ultraviolet (UV) radiation contributes to the aetiology of melanoma, but the precise mechanistic details are still unclear. The CDKN2A gene which is associated with familial and sporadic melanoma, encodes a tumour suppressor, p16. We have previously shown that in response to low doses of UV radiation the level of p16 increases, and that this correlates with a G2 delay. Here we report that in melanoma cell lines which do not express p16, or express a mutant p16, no G2 delay is observed in response to UV. The loss of functional p16 also correlates with an increase in DNA damage as judged by increased numbers of bi- and multinuclear cells and cells containing 1-2 micronuclei following UV irradiation. This work provides a further link between UV radiation, CDKN2A and melanoma, suggesting that the functional inactivation of CDKN2A disrupts a p16-dependent G2 cell cycle checkpoint, thus contributing to the development of this neoplasm.

Cell Cycle↗
PubMedDate not supplied

Cold-shock and the Mammalian cell cycle.

Progression through the cell cycle is temperature sensitive, but the relationship is not straightforward. In culture, many types of mammalian cells fail to undergo the G(2)/M transition after cooling from 37 degrees C to 16-20 degrees C (moderate hypothermia). However, progression through G(1) and S is not blocked at these temperatures, nor is progression through mitosis in cells cooled after they have become committed to the division process. Thus, at least one pathway is present during G(2)-but not during G(1), S or mitosis-that is selectively disrupted at or below a critical temperature. As a result, a prolonged (24-48 hr) exposure to moderate hypothermia can be used to enrich cultures for G(2) cells. A brief (1 hr) exposure to severe hypothermia (4-10 degrees C) is also reported to induce a high degree of mitotic synchrony (up to 80%) in some mammalian cultures. Although the mechanism behind this synchronization remains vague, it may involve a cell cycle checkpoint, triggered in response to the cold shock, that transiently inhibits the G(1)/S transition.

Animals↗
PubMed1996

Hyperthermia and paraquat-induced G1 arrest in the yeast Saccharomyces cerevisiae is independent of the RAD9 gene.

Mutants of the RAD9 gene of Saccharomyces cerevisiae are defective in cell cycle checkpoint arrest in G1 and G2 after treatment with DNA-damaging agents. It is demonstrated that the pronounced G1 arrest observed in yeast after hyperthermia treatment or exposure to paraquat-generated superoxide radicals does not depend on a functional RAD9 gene. For both types of treatments, the mechanism of cell cycle arrest must be considered different from the activation of the DNA damage checkpoint response.

DNA Damage↗
PubMed2000

Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity.

The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by gamma-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint.

Amino Acid Sequence↗
PubMed1995

DNA polymerase epsilon links the DNA replication machinery to the S phase checkpoint.

Inhibition of DNA synthesis induces transcription of DNA damage-inducible genes and prevents mitotic entry through the action of the S phase checkpoint. We have isolated a mutant, dun2, defective for both of these responses. DUN2 is identical to POL2, encoding DNA polymerase epsilon (pol epsilon). Unlike sad1 mutants defective for multiple cell cycle checkpoints, pol2 mutants are defective only for the S phase checkpoint and the activation of DUN1 kinase necessary for the transcriptional response to damage. Interallelic complementation and mutation analysis indicate that pol epsilon contains two separable essential domains, an N-terminal polymerase domain and a C-terminal checkpoint domain unique to epsilon polymerases. We propose that DNA pol epsilon acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks.

Alleles↗
PubMed2005

CtIP, a candidate tumor susceptibility gene is a team player with luminaries.

CtIP is a nuclear protein conserved among vertebrates that was discovered as a cofactor of the transcriptional corepressor CtBP. CtIP also interacts with the tumor suppressors such as BRCA1 and the pRb family members through binding sites that are frequently mutated in human cancers. CtIP is a target for BRCA1-dependent phosphorylation by the ATM kinase induced by DNA double strand breakage. CtIP plays a role in DNA-damage-induced cell cycle checkpoint control at the G2/M transition. Homozygous inactivation of the Ctip gene causes very early embryonic lethality during mouse development. The Ctip(-/-) embryo cells are arrested in G1 and do not enter S phase. Depletion of Ctip in established mouse embryo fibroblasts arrests cells in G1 and results in an accumulation of hypophosphorylated Rb and the Cdk inhibitor p21, suggesting that CtIP is also a critical regulator of G1/S transition of the cell cycle. The Ctip gene contains a mononucleotide (A9) repeat and one of the alleles is mutated at a high frequency in colon cancers with microsatellite instability. The Ctip(+/-) mice develop multiple types of tumors suggesting that haploid insufficiency of Ctip leads to tumorigenesis. Among the various tumor types observed in Ctip(+/-) heterozygous mice, large lymphomas are prevalent. Recent studies raise the possibility that Ctip may itself be a tumor susceptibility gene and suggest that it might be important for the activities of tumor suppressors BRCA1, pRb family proteins and Ikaros family members.

Alcohol Oxidoreductases↗
PubMed1998

Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition.

Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1). We provide evidence that in addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21-/- mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls.

Cell Nucleus↗
PubMed2002

Ectopic D-type cyclin expression induces not only DNA replication but also cell division in Arabidopsis trichomes.

Although the mechanisms controlling the two cell-cycle checkpoints G(1)-S and G(2)-M are well studied, it remains elusive how they are linked in higher eukaryotes. In animals, D-type cyclins have been implicated in the control of cell-cycle progression in mitotic as well as in endoreduplicating cells. By contrast, we show that the expression of the D-type cyclin CYCD3;1 in endoreduplicating Arabidopsis trichome cells not only induced DNA replication but also cell divisions.

Arabidopsis↗